Plant extract based composition useful for leishman IA promastigotes

ABSTRACT

This invention relates to a culture medium for cultivation of  Leishmania  promastigotes, comprising inorganic salts, organic constituents and peptone/hydrolystate and at least one plant extract.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the United States national phase of InternationalApplication No. PCT/IN2014/000270 filed Apr. 25, 2014, and claimspriority to Indian Patent Application No. 1233/DEL/2013 filed Apr. 26,2013, the disclosures of which are hereby incorporated in their entiretyby reference.

FIELD OF THE INVENTION

This invention relates to a plant extract based culture medium usefulfor Leishmania promastigotes. More particularly, the present inventionrelates to a culture medium having composition essentially comprisingplant extract useful for growth of Leishmania promastigotes, a parasitiehaemoflagellate of human interest.

PRIOR ART OF THE INVENTION

Leishmaniasis is a disease spread over tropical and subtropical regionof 88 countries (W.H.O., 1998. Leishmania and HIV in grid lock.WHO/UNAIDS report. Geneva: World Health Organization. 15-25). Amongwhich, 16 are developed countries, 72 developing countries and 13 leastdeveloped (Desjeux P, 1996. Leishmaniasis: public health aspects andcontrol. Clin. Dermatol. 14: 417-423). It is caused by members ofhemoflageilate protozoan parasites of the genus Leishmania and istransmitted by the bite of female phlebotomine sandflies. The parasiteundergoes a digenic life cycle between a nonmotile intracellularamastigote stage parasitizing the mammalian phagocytic cells and aflagellated, motile promastigote stage in the midgut of its sandflyvector (Adler S, 1964. Leishmania. Dawes B, ed. Advances inParasitology. Volume 2. New York: Academic Press, Inc., 35-96). Asimilar promastigote form develops when parasites are cultured incell-free medium. Leishmania spp. do not have heme-biosynthetic pathwayand require exogenous heme as essential growth factor fromserum/blood/blood products present in immediate environment for growthand multiplication (Chang, C. S. and Chang, K. P. 1985. Heme requirementand acquisition by extracellular and intracellular stages of Leishmaniamexicana amazonensis. Mol. Biochem. Parasitol. 16: 267-276; Soteriadou,K., Papavassiliou, P., Voyiatzaki, C., Boelaert, J., 1995. Effect ofiron chelation on the in-vitro growth of Leishmonio promastigotes. J.Antimicrob. Chemother 35, 23-29). Therefore, a medium containing hemesubstances e.g. blood/a blood product is required for culturingLeishrnotvo promastigotes.

Since the beginning of 20th century, cultivation of the Leishmoniodeveloped with NNN medium (the Novy-Mc Neal (1904) medium, as modifiedby Nicolle (1908)), which consist blood agar slant inoculated withinfected materials. Till then, the modifications in NNN mediumespecially in the basal phase are being carried out, which involvereplacement of the bacto-agar with richer nutrient agars (i.e. Difcoblood-agar, Walton B C, Shaw J J and Lainson R, 1977. Observations onthe in vitro cultivation of Leishmonio braziliensis. J. Parasitol.63:1118-1119; brain heart infusion agar, Marin F, De Lomas, J G,Penarrubia M P G and Penaluer J. 1982. Cultivation of Leishmonio:comparison of different media for promastigotes cultivation. Ann. Trop.Med. Parasitol. 76: 607-613; various tissue extracts, or serum plasmaWeinman, 1960. Trans. Roy. Soc. trop. Med. Hyg., 54, 180) has also beentested.

Several semi-defined/defined and biphasic/monophasic media have beendeveloped for the cultivation of different species or sub-species ofLeishmonio parasites (Steiger R F and Steiger E., 1976. A defined mediumfor cultivating Leishmonio donovoni and L braziliensis. J. Parasitol.62: 1010-1011; Steiger R F and Steiger E, 1977. Cultivation Leishmoniodonovoni and L. Braziliensis in defined media: nutritional requirements.J. Protozool. 24(3): 437-441; Berens, R. L. and Marr, J. J. 1978. Aneasily prepared defined medium for cultivation of Leishmonio donovonipromastigotes. J. Parasitol. 64: 160-167; Dumon H, Manoury B, Quilici Mand Ranque J, 1981. Growth of Leishmonio infantum in continuous culture.C R. Seances Soc. Biol. Fil. 175 (1): 82-86; Chaudhary, G., Chatterjee,T. K. and Banerjee, A. B., 1982. Growth factor requirements for in vitrogrowth of Leishmonio donovoni, Indian J. Med. Res. 76: 157-163.; LaFuente C, Saucedo E and Urjel R, 1983. A liquid modification of DifcoBlood Agar medium for cultivation of Leishmonio. Trans. R. Soc. Trop MedHyg. 77 (6): 882; Melo N M, De Azevedo H P, Roitman I, Mayrink W, 1985.A new defined medium for cultivating Leishmonio promastigotes. ActaTrop. 42(4): 137-141; Chaudhary G, Ghoshal K, Sen S, Pal S and BanerjeeA B, 1986. Nutrition of Leishmonio donovoni donovoni: Growth in new semidefined & completely chemically defined media. Indian J. Med. Res. 84:461-468; O'Daly J A and Rodriguez M B, 1988. Differential growthrequirements of several Leishmonio spp. in chemically defined culturemedia. Acta Trop. 45(2): 109-126; Gupta A K and Saran R, 1991. In vitromaintenance of Leishmonio donovoni promastigotes in a cheap, serum-free,hemin-based, autoclavable culture medium. J. Commun. Dis. 23: 276-2 77;Mc Carthy-Burke C, Bates P and Dwyer D M, 1991. Leishmonio donovoni: useof two different, commercially available, chemically defined media forcontinuous in vitro cultivation of promastigotes; Exp. Parasitol. 73(3): 385-387; Ozbilgin A, Ozbel Y, Alkan M Z, Atambay, M, Ozcel M A,1995. Cultivation of Leishmonio sp. in nutrient broth. J Egypt SocParasitol 25: 437-41; Lozano J, Campos M, Manas I and Diaz V, 1996.Semi-defined medium for cultivating Leishmanio infantum promastigotes.Ars. Pharm. 37 (3): 563-567; Limoneu M E, Balcioglu C, Yereli K, Ozbel Yand Ozbilgin A, 1997. A new experimental in-vitro culture medium forcultivation of Leishmanio species. Journal of Clinical Microbiology. 35(9) 2 430-2431; AN et al., 1998; Merlen T, Sereno D, Brajon N, Rostand Fand Lemesre J L, 1999. Leishmanio spp: completely defined medium withoutserum and macromolecules (CDM/LP) for continuous in-vitro cultivation ofinfective promastigotes forms. Am. J. Trop. Med. Hyg. 60: 41-50; MazyadS A and. Abdel-Aziz A, 2003. Peptone-liver (P-L) a new culture mediumfor the Egyptian Leishmanio major. J. Egypt. Soc. Parasitol. 33 (1):41-45.

Presently, all the basal culture media either semi-defined/defined,formulated or based upon commercial tissue culture media needsupplementation of animal-derived products such as blood (chiefly pooledfresh rabbit blood at concentrations ca. 2.5 to 30%) or FCS (at ca. 5 to30%), or adult serum (Evans D A, 1986. An inexpensive easily availablereplacement for foetal calf serum in media for the in vitro cultivationof Leishmanio spp. 7 Parasitenkd 72: 567-572), or blood products i.e.hemin or hemoglobin (Aljeboori T I, 1979. A simple biphasic mediumlacking whole blood for culturing Leishmanio spp. Trans. R. Soc. Trop.Med. Hyg. 73 (1): 117; Gupta A K and Saran R, 1991. In vitro maintenanceof Leishmanio donovani promastigotes in a cheap, serum-free,hemin-based, autoclavable culture medium. J. Commun. Dis. 23: 276-277;Bhattacharya, J., Mukharjee, H., Das, D. C. and Hati, A. K. (1994) Asimple medium without blood modified for successful isolation andcultivation of LD bodies. Indian J. Med. Res. 99: 171-172; Gupta A K,Narayan S and Saran R, 1995. Blood-based autoclavable medium forin-vitro cultivation of Leishmonia donovoni promastigotes. Indian J.Comp. Microbiol. Immunol. Infect. Dis. 16 (3-4): 144 145; Agarwal M andJain A, 1996. New blood free biphasic medium with haemoglobin forcultivation of Leishmonia donovani promastigotes. Indian J. Exp. Biol.34 (12): 1233-1236) or may also involve bovine serum albumin, peptone,infusion, extract.

The culture media designed specifically for Leishmanio cultivationcontain either fetal calf serum (FCS) or blood lysate as one of theiressential ingredients (Hendricks L D and Childs G E, 1980. Presentknowledge of the in vitro cultivation of Leishmonia. The in vitroCultivation of the pathogens of Tropical Diseases. Tropical DiseasesResearch Series 3, Schwabe and Co. A G. Basel, pp. 251-272; Lemesre J L,Darcy F, Kweider M, Capron A, Santoro F, 1988. Requirements of definedcultivation conditions for standard growth of Leishmonia promastigotesin vitro. Acta Trop 45: 99-108; Ozbilgin A, Ozbel Y, Alkan M Z, Atambay,M, Ozcel M A, 1995. Cultivation of Leishmonia sp. in nutrient broth. JEgypt Soc Parasitoi 25: 437-41). Several serum-enriched tissue culturemedia; initially formulated for mammalian cells cultivation (viz.Eagle's minimum essential medium, TC-199 and Dulbecco's medium, BerensR. L, Brun R and Krassner S M, 1976. A simple monophasic medium foraxenic culture of hemoflagellates. J. Parasitol. 62:360-365; Enders B,Brauns F and Zwisler O, 1977 Biochemical and technical considerationregarding the mass production of certain parasitic protozoa. Bull. W. H.O. 55: 393-402.; Childs G E, Foster K A and Mc Roberts M J, 1978. Insectcell culture media viz. Schneider's, Grace's and Mitsuhashi's medium,have now also been used successfully to cultivate several old and newworld Leishmanio species. Reports suggested that supplemented insectcell culture media gave higher yields for wider range of Leishmanio spp.(Hendricks L D and Childs G E, 1980. Present knowledge of the in vitrocultivation of Leishmanio. The in vitro Cultivation of the pathogens ofTropical Diseases. Tropical Diseases Research Series 3, Schwabe and Co.A G. Basel, pp. 251-272) and Mitsuhashi's medium with 20% FCS supporteda particularly different stock of L. b. Braziliensis (Walton B. C, Shawj j and Lainson R, 1977. Observations on the in vitro cultivation ofLeishmania braziliensis J. Parasitol. 63:1118-1119. Hendricks L D andChilds G E, 1980. Present knowledge of the in vitro cultivation ofLeishmanio. The in vitro Cultivation of the pathogens of TropicalDiseases. Tropical Diseases Research Series 3, Schwabe and Co. A G.Basel, pp. 251-272).

Rabbit's blood is the most common and suitable additive of basal media.

However, the quarantine free animals through antibiotics-free diet toget the highest quality of rabbits' blood regularly/routinely forcontinuous supply of pooled rabbits' blood (to minimized the chance ofanti-leishmanial effect, if present in any rabbit blood) for properperformance of the prepared culture medium is also a recurringexpenditure resulting high cost of blood-based medium. The collection ofblood from heart in sterile form from rabbits (Evans D A, 1986. Aninexpensive easily available replacement for foetal calf serum in mediafor the in vitro cultivation of Leishmanio spp. Z Parasitenkd 72:567-572) is also very expensive.

Though, FCS is an excellent supplement and replacement of blood in cellculture media that provides lots of defined and undefined essentialcomponents, yet, not acceptable because of killing the animals toprovide serum. Moreover, FCS is highly expensive (high-quality FCS cancost up to $1000 per liter) and its reliable supply is difficultespecially in developing countries (Chang K P and Hendricks L D, 1985.Laboratory cultivation and maintenance of Leishmanio: Chang, K. P. andBray, R. S. (eds) Human Parasitic Diseases, Leishmaniasis. Amsterdam-NewYork-Oxford; Elsevier Science Publishers, 213-247; Newman C. 2003.Serum-free cell culture—the ethical, scientific and economic choice.Biomed. Scientist: 941-942). Serum can also harbour hazardouscontaminants such as viruses, bacteria, prions, and mycoplasma (NewmanC. 2003. Serum-free cell culture-the ethical, scientific and economicchoice. Biomed. Scientist: 941-942). Above all, the increasing concernsabout animal suffering inflicted during collection add an ethicalimperative to move away from the use of serum wherever possible.

Various researches have been conducted to replace FBS with bovine serumalbumin or a mixture of purine bases, vitamins, large concentrations ofcertain amino acids, hormones, hemin, hemoglobulin and, more recently,even human and animal urine (Ali S A, Iqbal J, Ahmed B and Masoom M,1998. A semi-synthetic fetal calf serum-free medium for in vitrocultivation of Leishmania promastigotes. Am. J. Trop. Med. Hyg. 59:163-165; Armstrong T C and Patterson J I, 1994. Cultivation ofLeishmania braziliensis in an economical-serum-free medium containinghuman urine. J. Parasltol. 80 (6):1030-1032; Chaudhary G, Ghoshal K, PalS and Banerjee A B, 1986a. A new medium for large-scale production ofLeishmania donovani promastigotes for biochemical studies. Indian J.Med. Res. 84:457-460; Merlen T, Sereno D, Brajon N, Rostand F andLemesre J L, 1999. Leishmania spp: completely defined medium withoutserum and macromolecules (CDM/LP) for the continuous in-vitrocultivation of infective promastigotes forms. Am. J. Trop. Med. Hyg. 60:41-50; Pal J K and Purandare M J, 2001. Dose-dependent differentialeffect of hemin on protein synthesis and cell proliferation inLeishmania donovani promastigotes cultured in vitro. J. Biosci. 26:225-231; Shamsuzzaman S M, Furuya M, Korenaga M, Imamura K andHashiguchi Y, 1999. Use of urine samples from healthy humans, nephritispatients or other animals as an alternative to foetal calf serum theculture of Leishmania (L.) donovani in vitro. Ann. Trop. Med. Parasitol.93 (6): 613-620). These media lacks whole serum as an ingredient, but itmay not be entirely free of serum/blood/animal-derived products. Thesemedia may have defined quantities of purified obligatory growth factorsof animal origin such as hemin/hemoglobin, serum albumin and otherorganics like peptone/extract/infusion of meat, liver, brain, heartincluding milk of animal origin to enrich the semi-defined medium. Thechances of contamination either through animal-derivedpeptone/extract/infusion, or through hydrolyzing enzymes, if store inBSA, are also there. Being un-economical and un-ethical, involvingcomplex procedures and laborious preparation, the animal product basedculture media containing FCS/blood/blood products are still used due tothe lack of proper alternatives/due to significant impediments in priorart. To overcome the potential problems of the use of serum/fresh blood,a need exists for products originating from plant sources that cansupport multiplication of Leishmania promastigotes.

OBJECTS OF THE INVENTION

It is therefore an object of this invention to propose a culture mediumfor Leishmania which uses plant derived natural components.

It is a further object of this invention to propose a culture medium forLeishmania, which uses easily available materials and is easy toprepare.

Another object of this invention is to propose a culture medium forLeishmania, which has sufficient storage durability.

Yet another object of this invention is to propose a culture medium forLeishmania, which is cost-effective.

A further object of this invention is to propose a culture medium forLeishmania, which is user-friendly and eco-friendly.

A still further object of this invention is to propose a culture mediumfor Leishmania, which is free from the risks involved with the use ofanimal products.

These and other objects and advantages of the invention will be apparentfrom the ensuing description.

DESCRIPTION OF THE INVENTION

Thus according to this invention is provided a plant extract basedculture medium useful for growth of Leishmania promastigotes.

In accordance with this invention, is provided a plant based culturemedium for in-vitro culture of Leishmania promastigotes, a parasitichaemoflagellate. The culture medium and medium supplement of plant'sextract is a substitute for FCS/blood and other enrichment products ofanimal origin in basal medium, and is serum-free as well as totallyproduct free.

In present invention, the best approach to culture Leishmania parasitesmainly based on nutrition requirements considering the facts that apartfrom blood, adult female sand flies also take “plant diet”.

After infective bites, since the developmental cycle of Leishmaniapromastigotes in sand fly is permanently restricted to alimentary canal,they exposed to the ingested tissues/sap of the various plants uponwhich the sand flies feed. Hence, the different plants' food compositionwill not only affect the sand flies, but there is probability that aconsiderable number of plants' food of local flora must play a crucialrole in survival/propagation of Leishmania parasites present in the gutof naturally infected sand fly to continue/increase the potential ofLeishmania transmission by bite.

Leishmania promastigotes require hemin as obligatory growth factor asthey lack the enzymes i.e. aminolevulinate dehydratase andporphobilinogen deaminase which facilitate tetrapyrrole synthesis(Salzman T A, Stella Ana M., Xifra E A W de, Battle A M del C, Docampo Rand Stoppani A O M, 1982. Porphyrin biosynthesis in parasitichemoflagellates: functional and defective enzymes in Trypanosoma cruzi.Comp. Biochem. Physiol. 72B: 663-667).

It is also important that heme should be provided in optimumconcentration. Studies revealed that normal protein synthesis andproliferation of promastigotes occurred at optimum concentration (10 μM)of hemin but its inhibition at higher concentration (50 μM) (Pal J K andPurandare M J, 2001).

Another obligatory nutritional requirement of Leishmania promastigotesis purine, as they are unable to synthesize purine de novo glycine,serine and formate, and require an exogenous source of purine for theiractive growth.

In addition, most of the parasitic protozoa including Leishmania requirewater-soluble vitamins for their better growth.

Further, since all plants even of same species do not show similarresponse owing to differ in their biochemical and physiologicalconstitution due to climatic, edaphic and other factors and hence, inpresent invention, the potentiality of different plant's extract forcultivation of Leishmania promastigotes have been explored which was notknown so far. In present invention, it has been found that plant'sextract of a considerable number of plants of local flora supportedproliferation of Leishmania promastigotes in long-term continuoussuccessive sub-passaging. The invention provides novel culture mediacomprising plant-derived natural additive as essential growth factorsfor culturing promastigotes. No document is available in literaturerelated to use of plant's extract as supplement/additive in culturemedia and for different related applications of promastigotes. The sueof plants' extract is an extremely economical alternative as mediumadditive, which can be prepared in bench-scale or in bulk fromnon-exhaustable, easily available nautra resources. Hence, in the fieldof culture media preparation, an attractive goal is achieved forhemoflagellate cultivation employing plant's extract as a mediumsupplement in medium having peptone and plant origin.

The plants used in the present invention are selected from thefollowing:

Plant Code Botanical Name Family 5 Citrus maxima Rutaceae 16 Thevetiaperuviana Apocynaceae 26 Croton bonplandianum Euphorbiaceae 29 Benincasahispida Cucurbitaceae 15 Dolichos lablab Papilionaceae 23 Calotropisprocera Asclepiadaceae 27 Colocasia esculenta Araceae 28 Amorphophalluspaeoniifolius Araceae 24 Coccinia cordifolia Cucurbitaceae 13 Amaranthusdubius Amaranthacesae 42 Citrus aurantifelia Rutaceae 41 Cucurbita pepoCucurbitaceae 1 Sesbania sesban Papilionaceae 2 Solanum melongenaSolanaceae 44 Abutilon indicum Malvaceae 48 Trianthema portulacastrumAizoaceae 43 Basella alba Basellaceae

A novel (serum-free as well as totally animal product free) culturemedium has been developed for in-vitro culture of Leishmaniapromastigotes—a parasitic hemoflagellate of human interest.

Promastigotes can propagate in claimed medium during long-termcontinuous successive sub-passaging.

In one embodiment, the culture medium comprise inorganic salts e.g.sodium chloride, potassium chloride, potassium dihydrogen phosphate,calcium chloride, and organic mixture e.g. glucose, yeast extract andpeptone of plant source.

The plants' extract serve as an additive to provide the obligatorygrowth factor(s) for Leishmania promastigotes and their multiplicationin-vitro in continuous manner.

Extract of various plants that exhibited propagative property, belongsto different families and possess different characteristics, haveprovided more choice in screening of plants to get (raw material)plants' extract easily and economically in different seasons and places,and for better acceptability and performance.

Culture medium as well as few plants' extract retained their capabilityto promote propagation even after autoclaving.

Culture medium (having 1% agar) supplemented with plant's extract isalso capable and sensitive to promote cell growth from single cell tonegate clonal populations, especially if maintained in animal productfree medium.

Promastigotes (old/new isolates) can proliferate adequately in thisnutritionally changed growth environment without need ofsequential/special regimens for adaptation, prior to efficient long-termserial culture.

Promastigotes continuously sub-passaged in this medium when inoculatedin susceptible animal; parasites recovered from spleen suggestedretention of infectivity of promastigotes.

The complete medium can act as a serum/animal product free glycerolcryo-medium for preserving the culture from contamination with animaloriginated pathogens, which may incurred through use of animal productsuch as FCS/serum/infusion/extract/peptone etc.

Plants' extract either kept at least 3 years at −20° C. or 1 year at 4°C. or 6 months at room temperature, or even complete medium kept at 4°C. for at least 6 months exhibited luxuriant growth. Hence, the storagedurability is quite enough for convenient utility.

The mass production of medium supplement as well as its vacuum driedproduct can be achieved in industry at a low cost.

The culture system is user-friendly, eco-friendly and in compliance withregulations, to meet the ever-increasing needs of promastigotes as wellas for variety of related applications.

In the following description, a number of terms are used conventionallyin the field of cell culture media. In order to provide a clear andconsistent understanding of the specification and claims and the scopeto be given, the following definitions are provided.

The term “leishmanial” refers to a genus responsible for the diseaseleishmaniasis.

“Leishmaniasis” is a disease caused by protozoan parasites that belongto the genus Leishmania and is transmitted by the bite of certainspecies of sand fly (subfamily Phlebotominae).

“Promastigotes” is the flagellate stage of a trypanosomatid protozoan,as that of any of the Leishmania parasites.

“Amastigotes” is a non-flagellated phase in the life cycle oftrypanospme protozoans.

“Hemoflagellate” refers to a flagellate protozoan, such as atrypanosome, that is parasitic in the blood.

The term “cells” refers to individual cells as a whole/completeorganisms or promastigotes of Leishmania spp.

“Proliferate” refers to the property of one cell dividing into twoessentially identical cells or a population of cells increasing innumber (e.g., to reproduce).

“Propagation” refers to growing (e.g., reproducing via cellproliferation) cells outside of tissue or the body, for example, in asterile container such as a plastic/glass cell culture dish or flask.

“Cell culture” or “culture” refers to the maintenance/cultivation,growth, proliferation/propagation and/or differentiation of cells in anartificial in vitro environment.

“Cultivate” refers to the sustaining, propagating (growing) and/ordifferentiating of cells outside of tissue or the body.

The term “cultivation” refers to the maintenance of cells in-vitro underconditions favoring growth, differentiation or continued viability, inan active or quiescent state of the cells. “Cultivation” may be usedinterchangeably with “cell culture” or its probable synonyms such assub-culture or sub-passage. “Cultivation” may utilize a culture mediumas a source of nutrients and/or other factors helpful to propagateand/or sustain the cells.

“Capable of supporting the cultivation” refers to a condition thatallows the survival and proliferation and/or differentiation, ofisolated cells in culture.

“Passage”/“sub passage”/“sub culture” refers to the transfer of acertain portion of the cells from one culture vessel to another. It isunderstood that any time cells are transferred from one vessel toanother vials having fresh complete medium, dilution of cells willoccur.

“Passage number”/“sub passage number” refers to the number of times thecells in the culture have been subcultured or passaged.

“Serial passage” refers to the act of diluting and subdividing cellsinto multiple vessels when the cells have proliferated to a desiredextent. As cells are passaged from the primary culture vessel into asubsequent set of vessels, the subsequent cultures may be referred toherein “first passage” etc.

“Long-term cultivation” refers to the cultivation of cells for longperiods of time (months and/or years).

“Isolate” or “isolating” refers to separating and collecting cells fromtissue or the body (ex-vivo) in culture medium.

“Primary culture” refers to cells, tissue and/or culture where theisolated cells are placed in a first culture vessel with culture medium.The cells, tissue and/or culture may be sustained and/or mayproliferate, however, as long as the cells, tissue and/or culture remainin the first vessel the cells, tissue and culture are referred to as theprimary culture.

The term “ingredient” refers to any chemically defined or semi defined(in their composition) material, whether of chemical or biologicalorigin that can be used in cell culture media to maintain and/or promotethe growth and/or proliferation of cells.

The terms “component”, “nutrient” and “ingredient” can be usedinterchangeably and are all meant to refer to such compounds.

The “Infusion” is the outcome of material with desired chemicalcompounds. The ingredients, when admixed together in solution, form a“basal medium”.

The cell culture media/basal medium/medium supplement of the presentinvention are aqueous-based and comprise a number of ingredients in asolution of deionized distilled water to form a “basal medium”. Basalmedium developed by inventors has also been used in present culturesystem.

Basal salt's solution of this invention is enriched with the commonnutrients that comprise extracts of yeast cells (hereinafter “yeastextract”), peptone/infusion/extract of plant or animal origin andglucose to cultivate cells ex-vivo. This basal medium has beensupplemented additionally with certain plant's extract.

The phrases “cell culture medium” or “culture medium” (plural “media” ineach case) and “medium formulation” refer to a nutritive solution forcultivating cells and may be used interchangeably. Cell culture mediaprovide the nutrients necessary to maintain and grow cells in acontrolled, artificial and in-vitro environment. Cells cultivated inculture media metabolize available nutrients.

“Medium supplement” refers to nutritive compositions that help inmaintenance/growth/proliferation of cell.

“Complete medium” refers to combining or mixing or admixing ofsupplement(s) in a cell culture medium formulation. At least one plant'sextract is added to the basal medium to formulate the “complete culturemedia” of the present invention.

“Chemically semi defined medium” refers to a nutritive solution forculturing cells in which each component is not completely specifiable.

“Serum-free” medium means a medium that excludes serum of any type(e.g., fetal bovine serum, new born calf serum, horse serum, goat serum,etc.)

“Animal Product-free” medium means a medium that excludes any animalproduct such as serum, blood, hemin, hemoglobin, albumin, chicken embryoextract, peptone or extract or infusion of lean meat, brain, heart,liver, etc.

The term “culture vial/vessel” refers to a glass, plastic, or metalcontainer that can provide an aseptic environment for culturing cells.

The term “plant's extract” refers to a composition (comprising aconcentrated preparation of the component of a substance,) that may beformed by treatment of the substance/raw materials mechanically (e.g.,by blending, grinding, pressure treating) in water and that have notbeen treated with heat or additional chemicals or bio-chemicals. Thisparticular product is considered as a concentrated preparation of thecomponent of the plant's extract. It is a “non-hydrolyzed”product/extract, though may contain a minor amount of hydrolyzedproteins due to hydrolysis by endogenous enzymes.

The term “enzymatic digest” refers to a composition comprising aspecialized type of extract, namely one prepared by treating thesubstance to be extracted (e.g., plant components or yeast cells) withat least one enzyme capable of breaking down the components or thesubstance into simpler forms.

In this context, and for purpose of the present invention, the term“hydrolysate” may be used interchangeably with the term “enzymaticdigest”.

The term “non-animal derived”, “non-animal ingredient” or “derived fromnon-animal sources” refer to the origin of the compound of interest.Here, such non-animal source may include preparation or extraction ofmaterials of interest from members of plant kingdom e.g. plants andyeast cells.

“Plating” refers to spreading of cells on surface of solid medium.

“Clone” refers to a population of cells derived from a single cell bymitoses.

“Cryopreservation” refers to storage of cells in vapour/liquid Nitrogen.

“Infectivity” refers to establishment of infection in susceptibleanimals after in-vitro sub-passaging of organisms.

Materials and Methods

Selection of Plants:

The local herb and shrub plants (cultivated or wild, height <6″, some ofwhich having leaves at the soil level and stems not vertically erect)belonging to different families having different characteristicspreferably provide food/good shelter to sand fly in different seasonsand prevailing in the area of endemic villages of Bihar were selectedrandomly after site visit for the experiments.

Plants selection was based on the speculation that herbs and shrubs mayprovide suitable habitat for sand flies and probably give better chanceof feeding in different seasons. Some tree like plants (shrubs) werealso selected for preparation of plants' extract because these werecommon and branched with leaves at lower level (<6 ft) that may providethe same opportunity to vector. Plants were identified and coded.

Preparation of Plants' Extract:

The healthy plants free from insecticides/pesticides/fertilizers werecollected in humidified poly bags from different places in differentseasons during day time and brought to the laboratory immediately.

Plant's extract preparation was started within 2-3 hours aftercollection using stainless steel, glass, porcelain/china clay or plasticmade equipments taking care to avoid product contamination with othercomponents.

The plant's extract preparation procedure is as follows:

Soft leaves of each plant were washed thoroughly under running tap wateruntil dirt and epiphytes present on its surface removed and finallywashed thrice with doubled distilled water to remove tap water's traces.

Plants' leaves were blotted dried at room temperature, cut into smallpieces (if required) and added with chilled double distilled water in1:1 (w/v). The material was crushed using domestic applicant such aselectric operated domestic mixture-grinder, mortar-pestles at low and/ormoderate speed for 1-2 minutes avoiding generation of heat, and repeatedfor 6-7 times after cooling. The homogenate was strained throughdistilled water-washed muslin cloth to get a semi-clarified extract andallowed for freezing (at −20° C.) and thawing (at room temp.) for 2-3times that facilitate breakdown as well as formation and removal ofcold-insoluble material from non-hydrolyzed plant's extract. Thehomogenate was centrifuged at 4000-5000 rpm for 20-30 minutes andsupernatant was filtered through Whatman filter paper no. -1/Whatman-40filter paper. The filtrate was again centrifuged at 4000-5000 rpm for20-30 minutes. Cold-insoluble material need not be removed from thesolutions but may be removed for e.g., cosmetic reasons. After measuringthe volume, antibiotic (Gentamycin-100 μg/ml) was added.Supernatant/filtrate was sterilized by Millipore filter (size-0.22 μm),aliquoted into sterile vials and frozen at −20° C. or at desiredtemperatures for different durations for future use, if other wise notstated.

Parasites:

L donovani promastigotes (KA/PKDL isolates) were isolated in biphasicmedium (Brain heart infusion agar medium supplemented with 30% rabbitblood and having Locke's solution as overlay) from clinical materials ofan Indian KA patients who was treated and cured with anti-leishmanialtherapy.

Different species of Leishmania promastigotes such as L. tropica, Linfantum and L. major were procured from other research organizations.

These promastigotes were maintained in-vitro by weekly sub-passaging inthe biphasic medium having 10% rabbit blood or mono phasic medium having10% heat inactivated FCS.

Same isolates were used in different experiments if otherwise notstated.

Culture Media:

For initial screening of plant's extract, sub-passaging was done in ourinnovative medium i.e. LGPY (Locke's salt solution supplemented withglucose, peptone (animal or plant origin) and yeast extract) to see thelong-term propagative effects. After observing adequate propagation atleast up to 20^(th) sub-passage, RPMI (RPMI-1640 with glutamine, 25 mMHEPES Buffer and sodium bicarbonate, pH-7.4) and SIM (Schneider's insectmedium, pH-7.2) were compared with LGPY to see the suitability ofextract for long term continuous successive sub-passaging ofpromastigotes. Antibiotic (Gentamycin-100 μg/ml) was used in culturemedium.

Culture media were supplemented with 20% (v/v) of plant's extract, ifother wise not stated. The same media with heat inactivated FCS (10%,v/v) were taken as positive control and plain medium was taken asnegative control.

LGPY medium with heat inactivated FCS (10%, v/v) or BHI agar with 10%rabbit blood having Locke's solution as overlay was used to confirm therejuvenation/survival of promastigotes. Same batch of FCS was usedthroughout the experiments.

Culture and Sub-Passages Conditions:

Before inoculation in experimental media, the promastigotes weresub-passaged at least 3 times in the same medium supplemented with FCS(10%, v/v) to recognize the basal media. Promastigotes of log phaseculture were harvested and washed 3 times with sterile same plain mediumat 2500 rpm for 20 minutes at 4° C. to remove traces ofFCS/blood/hemin/hemoglobin in inoculum. The pallet was re-suspended insame cold medium and inoculated to get final concentration of 1×10⁵cells/ml in 5 ml screw caped glass culture vials having 990 μl of sameculture medium supplemented with 5, 10 and 20% plants' extract (inexperimental group), 10% FCS (in positive control) and plain culturemedium in negative control). Two culture vials in each group wereinoculated and were incubated at 24±1° C.

After inoculation of 5-7^(th) day, wet smear of each culture vial wasexamined microscopically to see the effect of plants' extract on killingand survival of promastigotes in experimental as well as in bothcontrols. Promastigotes viability was assessed in terms of their cellgrowth, flagellar motility and morphological changes (Habtemariam S,2003. Cytotoxic and cytostatic activity of erlangerins from Commiphoraerlongeriono. Toxicon. 41: 723-727), if promastigotes were active insub-cultured vials, 10 μl from each culture vial was transferred into 2corresponding fresh culture vials having same amount of medium. Ifmotile promastigotes were not found even then successive sub-passagingwas done further in corresponding culture medium till negative resultsobtained in following processes.

After inoculation, the rest suspension was centrifuged at 2000 rpm for20 minutes to remove maximum effect of plants' extract. The pallet wasdissolved in 50 μl LGPY without 10% FCS. A wet smear of this wasexamined microscopically for motile/sluggish promastigotes. If livepromastigotes were found after centrifugation, subculture in LGPY having10% FCS was not done, but If not, the rest suspension was inoculated inapproximately 1 ml of LGPY medium with 10% FCS or Brain Heart InfusionAgar medium having 30% pooled defibrinated rabbit blood having Locke'ssolution as overlay, incubated at 24±1° C. and wet smears of it wasexamined at every 2-3 days up to 2 weeks for the presence of livepromastigotes.

If all the promastigotes were found non-motile/degenerated duringmicroscopic examination, it was thought to be negative at particularsub-passage, Again the above procedure was followed.

These steps helped to confirm the killing/lethal effect if parasiteswere not rejuvenated and those viable promastigotes, which had escapedobservation during our microscopic examination. If the sub-culturefailed to show any motile one, it was considered as ill effect of thatplants' extract in particular medium at particular sub-passage.

Each experiment was done in duplicate vials.

In sub-passaging studies, growth density of promastigotes was determinedmicroscopically in per high-power field (HPF) taking equal amount ofcell suspension overlayed with equal sized cover-slip.

The invention will now be explained in greater details with the help ofthe following non-limiting examples:

Formulation of Basal LGPY Culture Media

The specific combinations of the ingredients, their concentration in thebasal media are shown in table—1.

TABLE 1 The LGPY base medium The LGPY base medium comprises inorganicsalts of Locke's salts type and organic mixture. Inorganic mixture, i.e.Sodium Chloride  8 g/l Potassium Chloride 200 mg/l Potassium dihydrogenphosphate 300 mg/l Calcium chloride 200 mg/l Organic mixture, i.e.Glucose  5 g/l Yeast extract  5 g/l Peptone/hydrolysate and Plantextract  10 g/l

Details of peptone/fusion of animal or plant origin are as bellow.

In one embodiment, Peptone of animal origin (i.e. HIMEDIA, RM 001/RM015/CR001).

In another embodiment, Soya Peptone (end product of enzymic, digestionof soyabean meal by papin—a plant origin based enzyme, HIMEDIA RM 007).

In another embodiment, HiVeg™ Peptone (an enzymic hydrolysate ofvegetable protein, HIMEDIA RM 001V).

In another embodiment, HiVeg™ Infusion Power (obtained from vegetableproteins under controlled conditions, HIMEDIA RM 188V).

In another embodiment, HiVeg™ Hydrolysate (prepared under controlledcondition by extracting vegetable proteins, HIMEDIA RM 023V)

All the ingredients were dissolved in 1000 ml double distilled water atpH 7.2-7.4 (pH was adjusted using NaOH). The solution was sterilized by0.22 μm Milli pore filter.

For sterilization by autoclaving, some of the ingredients were dissolvedseparately as below and mixed after autoclaving to form a complete basemedium.

The ingredients were taken in four flasks.

Flask—1 has NaCl, KCl, KH₂PO₄ and Peptone/Infusion, which dissolved in700 ml distilled water and pH (7.4-7.5) was adjusted with NaOH.

Flask—2 has CaCl₂ which dissolved in 100 ml distilled water.

Flask—3 has Glucose which dissolved in 100 ml distilled water.

Flask—4 has Yeast extract which dissolved in 100 ml distilled water.After autoclaving, all solutions of three flasks mixed together inlaminar airflow underneath the flame and antibiotic (Gentamycin-100μg/ml) was added:

Above basal medium was supplemented with aqueous plant's extract (about5-20%) as an obligatory additive like FCS/blood which is an inventivestep.

EXAMPLE-2

2A. Effect of Plants' Extract in Continuous Successive Sub-Passaging ofPromastigotes in FCS/Serum/Blood/Blood Products Free Medium i.e. LGPYHaving Peptone of Animal Origin

The hypothesis, that a considerable number of plant originatednon-hydrolyzed product herein plants' extract might be helpful inpropagation of promastigotes in long term continuous successivesub-culture, is verified through primary screening of plants' extract inFCS/serum/blood products free medium (if any). The plants have beenselected and the inventors used 10% and 20% of plants' extract forapproximation to optimum concentration for optimum best growth. Variousmammalian culture media such as RPMI 16P9, medium 858 and L-15, allsupplemented with 10% FCS, did not support the growth of L donovani formore than 2-3 subcultures, but in medium 199 with 10% FCS L donavanipromastigotes was reported to be sub-cultured for 2-3 sub-passages(Berens R L, Brun R and Krassner S M. 1976. A simple monophasic mediumfor axenic culture of hemoflagellates. J. Parasitol. 62: 360-365) andLb. panamensis (Hendricks L D, Wood D E and Hajduk M E. 1978.Hemoflagellates: Commercially available liquid media for rapidcultivation. Parasitology, 76: 309-316) for 17 weeks in the same mediumwith 30% FCS. Thus, a considerable number of sequential subcultures(usually at least 10) are required to establish a formulation as “growthmedium” rather than merely a “holding” or “washing” medium.

Before inoculation in experimental medium, the isolate was processed asdescribed previously in the heading culture and sub-passages conditions.

Plants' extract beneficial as a culture supplement in LGPY medium(possessed long-term proliferation promoting activity >15sub-passages/=positive control) are disclosed in table—2, but may not belimited to these specific plants only.

Some plant's extract that possessed efficacy <=negative control or 15sub-passages as a cell culture supplement in LGPY medium forpromastigotes are also disclosed in table—2.

TABLE 2 Effects of Plants' extract on promastigotes based on number ofsuccessive sub passages Effects of plant's extract On promastigotesDescription of effect/ (compared to Survival in No. Plant code no./control) of sub-passages Total No. Lethal/Harmful Non-motile or deformedcells 7, 14, 19, 20, 21, cells sustainment < negative 25, 32, 33, 34, 36Control (i.e. 0-4 sub-passages) 45, 46, 49/13 Short-term survival/ Cellssustainment >= 3, 4, 6, 8, 9, 10, 11, Propagation/ negative control/<=12, 17, 18, 22, 35, sustentacular 15 sub-passages 37, 38, 39/15Long-term survival/ Cells sustainment >= 1, 2, 5, 13, 15, 16,Propagation/ 15 sub-passages = 23, 24, 26, 27, 28, sustentacularpositive control 29, 30, 31, 40, 41, 42, 43, 44, 47, 48/21 Positivecontrol Cells sustainment >= At least 55 15 sub-passages Negetivecontrol 5

Some details and noticeable characteristics of plants' extract exhibitedlong-term proliferative effect are mentioned in table—3.

TABLE 3 Plants characteristics of which extract exhibited long termproliferative effect. Plant Plant Habitat Family Code Name C/W Age(A/P)(M/X) Acanthaceae 49 W A M Amaranthaceae 13 C A M Apocynaceae 16 C/W P XAsclepiadaceous 23 W P X Araceae 27 C A X 28 C/W A M Cucurbitaceae 24 WA M 29 C A M 33 C A M 41 C A M Euphorbiaceae 26 W A — Graminae 34 W A MIpomeaceae 35 W A M 36 W A M Lamiaceae 45 W A M + X 46 W A M Mimoceae 39W A M Malvaceae 31 C P M 40 C A M 44 W A M + X Papilionaceae 1 C A M 15C A M Piperaceae 30 C P M Rutacea 5 C P M 42 C P M 43 C A M Solanaceae 2C A M 32 W A M 37 C A M 38 C A M Umbellifereae 47 W A M 48 W A MA/P—Annual/Perennial 1, M/X—Mesophytic/Xerophytic, C/W—Cultivated/Wild.

Surprisingly, supplementation of one plant's extract (10-20%) in LGPYmedium exhibited successful proliferation of promastigotes at least upto 74 sub-passage and approximately ⅔ of tested plants' extract showedadequate proliferation at least up to 55 sub-passages.

Hence, this propagative property of plants' extract is evident in aconsiderable number of plants of different families/categories havingdifferent characteristics. Though, the propagative effect do notobserved only in particular families as all members of the same familydid not show similar response. Both, beneficial as well as harmfuleffect was observed in plant's extract of the same family, such asCucurbitaceae (4 out of 6 plants' extract), Solanaceae (4 out of 8plants' extract) and Malvaceae (3 out of 3 plants' extract) showedlong-term proliferative effect.

Results showed that the nature of plants (i.e. mesophytic, xerophytic)did not show any correlation with growth promoting ability of plants'extract. The selection of plants to get plants' extract as a productcould be based upon factors such as, their availability in differentseasons and places, easy cultivation in agricultural or bare land formass production of plant's extract for commercialization as well asbench scale production and collection of raw material in sufficientamounts and its processing under controlled conditions.

Some plants with soft, easily harvestable leaves (e.g. 1, 2, 13, 15, 16,27, 28, 29, 30, 40, 41, 43, 48) can be cultivated easily to get adequateamount of raw material.

Some plants are cultivated for use as a leafy vegetable (e.g. 13, 27,41, 43, 48) or to get their fruits (e.g. 5, 42) or fruits as a vegetable(e.g. 2, 15, 29, 40, 41) or corns for vegetables (e.g. 27, 28), andavailable in every seasons and place.

Some of the plants are perennial (t.g. 5, 16, 23, 42) or ornamental(e.g. 16, 31) and can serve whole year by providing abundant leaves.

The wild plants (e.g. 23, 24, 26, 44, 47) grow abundantly onuncultivated land around the peridomestic areas or in rural/sub urbanfarm, may also be available at free of cost.

During experiments of serial propagation of promastigotes, the inventorsdiscovered the non-hydrolysed plant derived extract supplement as a goodsubstitute of FCS/blood/its products in the medium which is cheap andreadily available. This indicated the possibility of presence of someyet uncharacterized growth factor (s) in plants' extract thatstimulate(s) proliferation of L. donovoni promastigotes. This ensuresthe presence of some active component(s) in plants' extract forreplacement of animal's product additives i.e. FCS/serum/blood or itsproducts.

Plant based natural biological fluid i.e. non hydrolysed plants' extractmight be a good source of heme containing substances like cytochromes,catalases/peroxidases as well as for purine and Water-soluble vitamin Bthat may fulfil the requirement of obligatory growth factors as well asgrowth promoting factors. In negative control, promastigotes died justafter few successive sub-passages if might not be supplied sufficientquantity of heme which is critical cofactor for many biologicalprocesses. In short and long-term propagative group, proliferation ofpromastigotes might be a result of combined effect of harmful as well asbeneficial components (whatever it is) present in the same extract invariable concentration.

Plant's product, herein, plant s extract can be a good alternative forreplacing FCS/serum/blood/blood-product in basal culture medium tosupport multiplication of promastigotes, which is an objective ofpresent study and is achieved through discovery that plant's extract ofa considerable number of plants of local flora has supporting effect inproliferation of Leishmonia promastigotes in long-term continuoussuccessive sub-passaging.

In another embodiment, plants which raw leaves' extract ensuredpropagative effect in serial culture were selected to ensure whetherextract of dried leaves will retain their obligatory growth factor(s)sufficient to replace FCS/blood or not. Aqueous extract from driedleaves of 3 plants' were prepared and dried. Then, medium wassupplemented with dried extract at 3 different concentrations i.e. 5, 25and 50 mg/ml (table 4).

TABLE 4 Confirmation of fertility properties of APF medium having 3extracts from dried leaves of 3 plants at 3 different conc. Results arebased on survival up to No. of SP. Survival in SP No. Solvents D.W.Conc. (mg/ml) 5 25 50 PE Code 5 2 2 1 15 3 2 1 16 3 5 3 Negative Control4 (only plain medium) Positive Control 10 continued (medium with FCS)

The results showed that obligatory growth factor lost during processing.Hence, our plants' extract preparation procedure to supplement mediumfor serial culture of Leishmario promastigote was a inventive step.

Use of extracts of aloe-vera plant leaves has already been suggested(PCT Application No. PCT/US99/09574). Unfortunately, test resultsdemonstrate that aloe-vera lea powder extracts do not possess activitysufficient to replace bovine pituitary extract (BPE) as a supplementbecause the extract lacks BPE's proliferation-promoting activity forimportant cell types (US Pat. Appli. No. 20060094114).

LGPY medium supplemented will vacuum dried extract obtained from rawleaves has also exhibited long term continuous successive sub passasing.

2B. Effects of Plants' Extract in Continuous Successive Sub-PassagingProcess of Promastigotes in Completely Animal Products Free Medium i.e.LGPY Medium to Ensure the Long Term Propagation

A number of semi-defined media, enriched with a variety of organics(including peptone, beef infusion, tryptose, liver extract, brain heartinfusion, yeast extract and, of course, fresh blood-chiefly pooledrabbit blood or FCS at concentrations ranged from 10-20%) have been usedsuccessfully to culture Leishmania promastigotes.

Taking lead from previous observations, to develop completely AnimalProduct Free culture medium for propagation of Leishmania promastigotesand their utility for different related applications, FCS/blood or itsproducts including peptone of animal origin of our trial medium wasreplaced by 4 different commercially available peptone/hydrolysate orinfusion of plant origin (as the composition depend upon source as wellas processing, and small change in medium enrichment is also among thedifferent factors that can have drastic effect on cell growth andpropagation) to satisfy nutritional contributions, and were supplementedwith 4 different plant's extract in 3 different concentrations (i.e. 20,10 and 5%) to confirm the fertility properties of the culture medium forlong-term continuous successive sub-passaging of promastigotes.

Adequate proliferation of promastigotes was exhibited at least 40sub-passages in all media i.e. LGPY medium having peptone/infusion andextract of plant's origin (table—5). This confirmed the survival ofpromastigotes in continuous successive sub-passaging in completely APFmedium. This reality was not known since starting from NNN. However, 3trial media having different peptone of plant origin did not supportlong-term propagation of promastigotes, even after supplemented with 10%FCS.

TABLE 5 Confirmation of long-term propagation of promastigotes in APFmedium having supplements of plant origin Survival in Sp No.Peptone/infusion PE code (conc. 5-20%) Control from plant 5 16 26 29 +ve−ve P 40 40 40 40 5 2 SP 40 40 40 40 4 3 HI 40 40 40 40 3 3 Si 40 40 4040 40 4 SP—sub passage; PE—plant's extract; P, HI & Sp—differentpeptone; Si—infusion

Ignoring the rate of proliferation, these sub-passage numbers are quiteenough to confirm that the medium supplemented with only plants'products can be used for long-term propagation of Leishmaniapromastigotes. Thus, in this culture system (vegetarian culture medium),components of plant's origin can satisfy all nutritional requirements ofpromastigotes for their growth and reproduction as similar to FBS/bloodor its products along with other enrichments of animal origin.

Thus, the present invention relates to methods for replacing all animalnatural-products such as FCS/serum/blood, or animal-derived productssuch as hemin/hemoglobin/peptides or infusion or extract etc. from plant(non-hydrolysed natural-products) along with plant-derived products i.e.peptides or infusion or extract. Such plant nutrients may be substitutefor any number of, animal-originated culture medium components orsubstituents, including but not limited to blood derived products in theculture media of the invention. Hence, a substantial useful progress hasbeen achieved by this new technological approach, which is fast,efficient, simpler and cost-effective.

The promastigotes proliferated well in plant-based medium indicatingthat plant extract's could be a source of heme containing substances, asthe components of basal medium did not have the iron source.

EXAMPLES-3

Microscopical Determination of Morphological Changes (MorphologicalStudies)

The morphological characteristics of the cultured promastigote formaccording to the invention were compared with the promastigote formcultured in conventional medium by light-optical microscopy becausecells tend to be more sensitive and fragile when cultured in animalproduct-free medium.

During continuous successive sub-passaging in this medium, wet-smear andGiemsa stained smear of log phase promastigote culture was examinedmicroscopically for presence of abnormal or atypical morphology. Theyretained their general morphological/cytological characteristics;indicated that plant extract as medium supplement is good for health andlong-term propagation of promastigotes.

EXAMPLE-4

Comparison for Long-Term Propagative Effect in Different MediaSupplemented with Different Plant's Extract:

To determine long term propagative effect of plants' extract in twocommercially available media (mammalian cell culture medium and insectcell culture medium), plants' extract (20%, v/v) were supplemented withSchneider's Insect medium (SIM) and RPMI (RPMI-1640 with glutamine, 25mM HEPES Buffer and Sodium bicarbonate) that have shown beneficialeffect with LGPY medium in long-term sub-passaging of promastigotes.

Antibiotics (100 μ/ml, Gentamycin or 100 IU/ml Pencillin and 100 μg/mlStreptomycin) were used in medium. The propagative effect was comparedwith LGPY having same plants' extract at same concentration. The mediumwith heat inactivated FCS (10%, v/v) were taken for positive control andplain medium as negative control. Complete LGPY medium (FCS 10%, v/v)was used to confirm the rejuvenation/survival of promastigotes, whichhave escaped observation during our microscopic examination. Eachexperiment was done in duplicate.

Two commercially available media (RPMI-1640 and SIM) and LGPY werecompared using 9 different plants' extract, all 9 plants' extractsshowed long term propagation in LGPY, whilst only 6 and 3 plants'extract out of 9 extracts' in SIM and RPMI-1640 respectively favor thelong term propagation at least up to 55 continuous successivesub-passages.

In present invention, 3 plants' extract favors the propagation at leastup to 55 continuous successive sub-passages in all the three culturemedia.

Results demonstrated that the composition of the basal medium isessential to the ability of the disclosed non-hydrolyzed plant extractsupplements to support the growth of, for example, Leishmaniapromastigotes.

Leishmania promastigotes are unable to synthesize purine (an essentialgrowth factor) de novo from glysine, serine and formate and require anexogenous source of purine for their growth. There is no purine base inboth commercially available media (i.e. RPMI-1640 and Schneider's insectmedium), and plant's extract alone may not, be able to fulfill thepurine requirement in these media. Hence, promastigotes died aftercertain subsequent sub-passaging with some plant's extract. Whereas, inLGPY, yeast extract and peptone are the good source of purine and thussupported the growth of promastigotes.

It has been reported that RPMI 1629, medium 858 and L-15 allsupplemented with 10% FCS did not support the growth of L. donovani formore than 2-3 subcultures and could only be cultured for 2-3 subpassagesin medium 199 with 10% FCS (Berens R L, Brun R and Krassner S M, 1976. Asimple monophasic medium for axenic culture of hemoflagellates. J.Parasitol. 62: 360-365), while L. 5. panamensis (Hendricks L D, Wood D Eand Hajduk M E, 1978. Hemoflagellates: Commercially available liquidmedia for rapid cultivation. Parasitology, 76:309-316) was sub culturedfor 17 weeks in medium 199 with 30% FCS.

Hence, different basal media are not equally good to supportproliferation with same/different plants' extract in this culturecondition, and ingredients of basal medium itself can affect much. Inthis way, our formulation of basal medium is found ideal/better growthpromoter in this culture condition.

The formulation according to the invention of basal medium is enrichedwith glucose, peptone and yeast extract. In fact, Leishmaniapromastigotes can grow in vitro without glucose, provided that aminoacids such as proline and glutamate are present in culture medium But,even then glucose was added as it was found that D-glucose andnon-esterified fatty acids stimulated promastigotes respiration andamastigotes transformation to promastigotes in vitro. Peptone and yeastextract contribute mainly for nitrogenous components such as aminoacids, nucleic acid bases, vitamins.

TABLE: 6 Effect of different plant's extract (n = 13) on growth ofpromastigotes based on No. of successive SP in 3 different culturemedia. Survival in No. of SP PE Code & control RPMI SIM LGPY 27 4 3 55 24 4 55 5 55 5 55 13 5 55 55 16 & 26 6 55 55 15, 28, 29 (9/3) 55 55 55Positive control 55 55 55 Negative control 4 3 5 9/4 9/6 9/9 PE—plants'extract, SP—sub-passage

EXAMPLE-5

Determination of Temperature Stability at 121° C. for 20 Minutes:

Autoclaving is the most economical, convenient, safe and reliablesterilization method that increases the practical acceptability formedium preparation. Hence, to confirm the thermal stability of medium aswell as plants' extract (found suitable in long-term maintenance ofpromastigotes in LGPY medium previously) were autoclaved at 121° C./15lb for 20 minutes and supplemented (20% v/v) in the same medium afterautoclaving followed by continuous successive sub passaging ofpromastigotes.

Interestingly, 2 (code—5 & 29, table—9) out of 21 exhibited adequategrowth in long-term continuous successive sub-passaging of promastigotesin ca. 50 sub-passages in-vitro cultures.

This ensured the growth factor of at least certain plants' extracts asheat stable at 121° C. for 20 minutes, might be due to its intrinsic aswell as extrinsic influences such as, presence of some other componentin plants' extracts, retention at particular pH etc.

Hence, at least few plants' extracts can be used after sterilization bymoist heat which are well-suited with respect to limited laboratoryfacilities and increase the practical utility and acceptability. Thefulfills one of the objects of invention i.e. to provide an autoclavablemedium supplement as well as basal medium to produce completelyautoclavable medium for long-term propagation of promastigotes.

TABLE 7 Thermal stability (at 121° C. for 20 min) of plants' extractused for promoting growth of Leishmania promastigotes. PE Code & controlno Total no. of PE Survival in SP. 40 1 0 42 1 3 1, 2, 16, 24, 26-28, 115 31, 43, 44, 47 30 1 7 41 1 8 23, 48 2 10 15 1 13 13 1 20 5, 29 2 40(21/2) Positive control 40 Negative control 5 PE—plants' extract,SP—sub-passage

EXAMPLE-6

Evaluation of Growth:

During experiments, adequate number of promastigotes was observed inLGPY medium supplemented with various plants' extract in long termcontinuous successive sub passaging of promastigotes. It was not knownwhether propagation of parasites in plants' extract supplemented mediumis quit enough/at par to FCS supplemented medium or not. Hence, it isworthwhile to illustrate the final density of particular cells in newmedium for different studies.

To clear this fact, experiment was done in 15 ml culture vial. LGPYmedium was supplemented with randomly selected extract (stored at −20°C. for 3 years) of 2 plants (perennial, 1 mesophytic & 1 xerophytic) in3 different concentrations (i.e. 20%, 10% and 5%). LGPY medium with FCS(10%) was taken as control. These media were inoculated with 1×10⁵cells/ml. Total number of cells was counted at every 24 hrs.

Supplementation of LGPY medium with another plant extract (code—16) at20% and 10% concentration, maximum growth crossed 1×10⁸ cells/ml (i.e.approx. 1.3×10⁸ cells/ml) on 7^(th) day. Hence, these concentrations arequite enough for mass/routine culture of promastogotes which can providelarge number of parasites in a short period of time (i.e. 7^(th) days)at extremely low cost. At 5% concentration of plants' extract, themaximum growth crossed 1×10⁷ cells/ml (i.e. 7.3×10⁷ cells/ml) on 7^(th)days. This may be used for routine maintenance of promastigotes.Survival of promastigotes was observed at least 7 weeks.

Supplementation of LGPY medium with another plant extract (code—5) at20% concentration, maximum growth crossed 1×10⁸ cells/ml (i.e. 1.1×10⁸cells/ml). The cell density was only little less at 10% supplementation(i.e. approximately 9.2×10⁷ cells/ml). Survival of promastigotes wasobserved at least 8 and 7 weeks, respectively. Hence, theseconcentrations are quite enough for mass/routine culture ofpromastigotes which can provide large number of parasites in a shortperiod of time (i.e. 6^(th) days) at extremely low cost. At 5%concentration of plants' extract, cell concentration was 5.3×10⁷cells/ml on 8^(th) day. Survival of promastigotes was observed at least2 weeks. Hence, culture at this concentration may be used for routinesub passasing of promastigotes.

Hence, it was found that number of parasites depends upon concentrationsand types of plant's extract. Different phases of promastigotes inculture medium were also clear to work on different phases ofpromastigotes.

Supplementation of LGPY medium with FCS (10%) exhibited 3.76×10⁷cells/ml on 12^(th) day and survival of promastigotes was observed atleast 7 weeks. Each experiment was done in duplicate.

EXAMPLE-7

Evaluation of Low Inoculum Size for Adequate Propagation ofPromastigotes:

It is also advisable that thresh hold of the inoculum size of cell/lowinoculum size of cell (here, <1×10⁵ promastigotes/ml) for propagation inthe newly developed culture medium need to be determined.

To determine the low inoculum size of promastigotes for adequatepropagation, LGPY medium having 10% concentration of one plants' extract(code—16) was inoculated with promastigotes at 3 different finalconcentration i.e. 1×10⁴, 1×10³ and 1×10² cells/ml. The total number ofcells was counted on 3 successive day (i.e. 7^(th), 8^(th) and 9^(th)day) starting from 7^(th) day, because maximum growth was observed on.7^(th) day with higher inoculum (i.e. 1×10⁵ cells/ml).

When LGPY medium supplemented with plant-extract was inoculated with1×10⁴ cells/ml, total number of cells/ml was approximately 3.6×10⁷cells/ml on 9^(th) day. When inoculated with 1×10³, total number ofcells/ml was around 1.5×10⁷ cells/ml on 9^(th) day. It was also observedthat plant extracts support cultivation of parasite even at very lowinoculums size (1×10² parasites/ml). When inoculated with 1×10², totalnumber of cells/ml was around 0.49×10⁷ cells/ml on 9^(th) day. Thesepromastigotes were very active.

Hence, LGPY medium with plant's extract is also quite sensitive topropagate promastigotes in adequate number and may be used in routine invitro maintenance of different isolates.

EXAMPLE-8

Determination of Effect of Storage Conditions (Temperatures andDurations):

While developing a new medium, it is important that basal medium, mediumadditive and complete medium should have adequate storage durability.

Plants' extract, complete medium and basal medium were stored atdifferent temperatures (i.e., −20° C., +4° C. and room temperature) fordifferent period of time (i.e., 4 years, 1, year, 9 months and 6 months)to evaluate the growth at different time period.

The complete medium were inoculated with equal number of promastigotes(i.e., 1×10⁵ cells/ml) to determine any noticeable/remarkable differencein density/population of promastigotes during continuous successivesub-passaging on 6-7^(th) days at different time period. The number wasevaluated microscopially by 40× objective taking equal amount (i.e., 15μl) of cell suspension on microscopic slide covered with equal size(18×18 mm) of cover glass.

The stability studies of the medium, complete medium and plant's extractproducts at different storage temperatures and durations demonstratethat the storage stability of the frozen plants' supplement are quitesufficient (at least 4 years) from the date of manufacture providingextra benefit to use for long duration like FCS. It could be stored at+4° C. for 1 year and at least 6 months at room temperature.Promastigotes were capable of undergoing long-term cultivation.

Since the basal medium as well as complete medium stored at roomtemperature for a quite considerable period of time (at least 6 months)did not lost the growth promoting properties of the plant's extractsaves valuable refrigeration space/reduced requirements for mediastorage and easy in transportation, if required.

TABLE 8 Effect of temperatures and durations on LGPY medium, plants'extract and LGPY medium with plants' extract. Effect of storageconditions (temp. & duration) Material (cells/ml) RT +4° C. −20° C.Growth PE 6 m 12 m  36 m Luxuriant LGPY 6 m Luxuriant LGPY + PE 6 mLuxuriant 9 m Poor 12 m  Nil PE—plant's extract, m—month, Luxuriant—>4.0cells/HPF, Poor—<10 cells/HPF

EXAMPLE-9

To Determine Adaptive Capability in New Growth Environment:

Some cells are very sensitive to physiochemical and nutritional changesand may take more than 8 consecutive passages for the adaptation (Hyq®CDM4NSO™). Sometimes special regimens are or may be required to adaptinsect cells (CHO and BHK cells to protein-free express media ofdifferent origin) from serum containing to serum free SF-3/SF-4 media:direct and gradual replacement (weaning).

Primary isolation of promastigotes was done in plant's extract (Code No.5, 16, 26 and 29) supplemented LGPY medium, and/or in blood basedbiphasic medium or FCS based monophasic medium. These isolates werecontinuously successively sub-passaged in corresponding medium asmentioned above.

Newly isolated or serially sub-passaged promastigotes from blood/serumbased medium were also inoculated into plant's extract based medium todetermine adaptive capability in nutritionally changed growthenvironment.

Promastigotes from blood/serum based medium when directly inoculatedafter three washing into the plants' extract based trial medium, grewvery well to the new growth environment, which indicated that prior toculture, sequential adaptation of promastigotes to grow in serum-freemedia is not required.

TABLE 9 To determine adaptive capability in new growth environmentClinical Isolation Sub-passaging material Isolate No medium mediumGrowth Splenic 8,8118 LGPY + PE NNN, LGPY + FCS Luxuriant aspirate &LGPY + PE Skin biopsy 247 NNN NNN, LGPY + FCS Luxuriant LGPY + PEPE—plants' extract

EXAMPLE-10

Determination of Colony Formation of Promastigotes on Solid Medium:

For cell cloning, agar plating is a very appropriate technique as beingsensitive and less laborious than competing methods. The colonyformation ability of Leishmania spp. on solid medium are exploited forimmunological (Handman E, Hocking R E, Mitchell G F and Spithill T W,1983. Isolation and characterization of infective and non-infectiveclones of Leishmania tropica. Molecular and Biochemical Parasitology 7,111-126), physiological (Simpson L, 1968. The Leishmania Leptomonadtransformation of Leishmania donovani: Nutritional requirements,respiration changes and antigenic changes. J. Protozol. 15: 201-207;Wagner K P and Krassner S M 1976. Leishmania tarentolae: streptomycinand chloramphenicol resistance of promastigotes. Exp. Parasitol. 39:222-233), genetic, drug sensitivity analysis (Pal S, Mandal A andDuttagupta S, 2001. Studies on Stibanate resistant Leishmania donovaniisolates of Indian origin; Indian J. Exp. Biol. 39 249-254;Bhattacharyya A, Mukherjee M and Duttagupta S, 2002. Studies onStibanate unresponsive isolates of Leishmania donovani. J. Biosci. 27:503-508) and virulence etc. For this a few medium has been evaluated fortheir efficacy (Keppal A D and Janovy J Jr., 1980. Morphology ofLeishmania donovani colonies grown on blood agar plates. J. Parasitol.66: 849-851; Iovannisci D M and Ullman B, 1983) High efficiency platingmethod for Leishmania promastigotes in semi-defined or completelydefined medium. J. Parasitol. 69: 633-636; Hill J O and Fahey J R, 1987.Leishmania spp; Agar plating as an alternative to limiting dilution andimpression smears for the enumeration of viable parasites in tissue: 63:108-111; Kar K, 1997. Folic acid the essential supplement to brain heartinfusion broth for cultivation and cloning of Leishmania donovanipromastigotes. Parasitology, 115: 231-235; Kar K, Mukerji K, Naskar K,Bhattacharya A and Gosh D K, 1990. Leishmania donovani: a chemicallydefined medium suitable for cultivation and cloning of promastigotes andtransformation of amastigotes to promastigotes. J. Protozool. 37:227-279). Growth as colonies on agar plates also offers some otherimportant advantages such as, generation of large number, of clones andtheir easy quantification on a single plate, determination of relativegrowth rates by direct visualization of colony size, enumeration ofviable parasites present in a tissue, transfer of clones en masse tosolid support (e.g. nitrocellulose membrane) for rapid screening,determination of nutrient requirements of cells, measurement of growthaffecting factors, toxicity testing etc. Hence, a colony formationefficacy in this novel medium was also determined.

To elucidate the colony formation from single cell on 1% agar base inLGPY medium supplemented with 3 different plants' extract (20% v/v), 2Petri plates of each were inoculated with 3 cells/plate which wasachieved by dilution of an original cell population down to 3 cells in50 μl. For positive control, 10% freshly obtained defibrinated rabbitblood was mixed with LGPY medium having agar (1%). The promastigotescolonies were counted and were compared with controls. The material ofeach colony was scratched by sterile loop and suspended in plain medium.Wet smear was examined under microscope for viability of cells. Coloniesdiameters were measured.

Whitish-mucoid viable colonies of 3 to 4 mm size were observed on10^(th) day in each plate. As it is a measure of the number of coloniesoriginating from single cells; it is clear that this medium (could alsopick up small inoculums to proliferate) is also very sensitive anduseful for the selection of single-cell colonies and has supported highcell densities with good viability and high productivity levels withoutthe use of animal-derived compounds. This formulation was tested with 3different plant's extract and was shown to generate comparable results,in term of clonal survival and growth. Thus, the plant's extractsupplemented medium supports Leishmania promastigotes clonal survivaland growth comparable to 10% FBS supplemented basal medium.

On this basis, this invention claims that serum-free medium iscomparable to serum-supplemented media. In order to address theregulatory needs, it is an object of the present invention to provide aculture medium for Animal Component—Free cloning that is capable ofsustaining growth of Leishmania promastigotes at very low cell densityconditions, and which is equivalent in cell cloning efficiency of mediahaving serum.

TABLE 10 Evaluation of colony formation (on 1% agar-base in LGPY with20% PE, inoculum 3 cells/plate) No. of colony on 10^(th) day PE code (n= 3) Plate I Plate II  5 2 3 16 3 2 26 4 2 Positive control (LGPY + 10%FCS) 2 3

EXAMPLE-11

Cryopreservation

Serum-particularly FBS—is used universally as a cryoprotective additiveto enhance the long-term cryostability of cells. If cells are grown inserum free medium, it is advisable to cryopreserve the cells in serumfree medium due to various reasons. The serum free freezing media suchas Promo cell's Cryo-SF M formulation, Hyclone Hyq® CDM4NSO™ formulation(Hyq® CDM4Retino™; Hyq® SFM4CHO™; Hyq® CDM4NS™; Hyq® CDM4m Ab™; Hyq®ADCF-mAb™) have been developed to cryopreserved the cells. In aserum-free cell culture system, the cells are cryopreserved in aserum-free freezing medium.

Use of serum-free cryopreservation media for protozoan parasites likeLeishmania promastigotes has received much less attention. Thus, thesearch for animal-source-free cryopreservation medium formulations hasbecome an important focus for culturist for long-term storage of cellbanks which is non-obvious.

After primary isolation, mass culture of the parasites were grown inLGPY medium having 10% four different plants' extract (n=4, Code No. 5,16, 26 & 29). Mid-log-phase culture of promastiogotes was harvested bycentrifugation for 20 minutes at 2500 rpm at 4° C. Number ofpromastigotes was adjusted to 2×10⁷ cells/ml in both experimental (LGPYmedium with 20-30% plant's extract) and control (LGPY medium with 20-30%FCS) group. Each group was supplemented with 16% Glycerol. Equal volumeof cold cryopretective medium was added drop by drop in cold parasites'suspension. The vials were cooled slowly and kept in liquid nitrogenfinally.

Cryopreserved promastigotes were examined after six months up to one andhalf year for their viability. Stabilities were thawed rapidly at 37° C.in water bath, further examined microsopically for their motility andsub-cultured aseptically into same kind of culture medium in which thecryopreserved promastigotes were grown before freezing. Sub culture wasexamined on 8^(th) days.

During wet smear examination, both motile and sluggish promastigotesexhibited luxurious growth on 8^(th) day in first and furthersub-passages.

In freezing test, the cells viability, frozen in the experimentalmedium, was good and for 1.5 year the result was similar to those grownand frozen in conventional medium. Furthermore, time for re-establishingcell division on thawing is also similar to conventional medium. Cellscryopreserved in this showed good performance after thawing insuccessive culture. Henceforth, it may act as a serum-free glycerolcryo-medium/cell freezing glycerol medium—serum free giving no chance ofcontamination of the culture through serum in the freezing medium.

Protozoa require cryoprotective agent when refrigerated at lowtemperature. Meryman H T, 1971. Cryoprotective agents. Cryobiology8:173-183). Glucose has been found as a natural cryoprotective agent tohelp counteract the osmotic shock and control changes in volume oftissue cells in Rana sylvatica (Devireddy R V, Barratt P R, Storey K Band Bischof, J C, 1999. Liver freezing response of the freeze-tolerantwood frog, Rana sylvatica, in the presence and absence of glucose. I.Experimental measurements. Cryobiology 38, 310-326) which is able totolerate freezing temperatures as low as −6° C. to 8° C. and durationsof freezing of 2 weeks or more with multiple freeze-thaw cycles. Acombination of 10% DMSO and 8% (v/v) glucose has been used to preservestrains of Deuteromycetes (Dietz A and Currie S, 1996. Actinomycetes, inMaintaining Cultures for Biotechnology and Industry (Hunter-Cevera, J.C. and Belt, A., eds), Academic Press, Inc., San Diego, Calif., pp.85-99).

Sucrose and trehalose (Beattie G M, Crowe J H, Lopez A D, Cirulli V,Ricordi C and Hayek A; 1997. “Trehalose: a cryoprotectant that enhancesrecovery and preserves function of human pancreatic islets afterlong-term storage.” Diabetes 46(3): 519-23; Eroglu A M J, Russo R,Bieganski A, Fowler S, Cheley H B and Toner M, 2000. “Intracellulartrehalose improves the survival of cryopreserved mammalian cells.” NatBiotechnol 18(2): 163-167) are often used in combination with DMSO asadjuncts in freezing media to increase the recovery of viable cells.These large mol. wt. substances protect the cell by facilitatingintra-cellular dehydration through diffusion of water out of the cellvia osmotic forces and inhibition of ice-crystal formation. At Eli Lilly& Company, the freezing medium composed primarily of a plant derivedpeptone and 10% (v/v) synthetic glycerin.

Our basal medium has glucose and other large-molecular-weight membranenon-permeating solutes like such as sucrose, trehalose, raffinose, etc.may come from plant's extract. Veg peptone like soya peptone has highcarbohydrate (total 24%) e.g. raffinose, sucrose and various otherreducing sugars and yeast extract also has about 17.5% totalcarbohydrate.

It is clear that this medium appears to provide measure of protectionduring freezing and thawing as judged by viability and re-growthpotential of promastigotes. Cryoprotective nature of the medium might bedue to collective effects of known cryoprotectants present in the mediumas medium supplements. However, this medium allows the storage of L.donovani isolates in liquid nitrogen for considerable periods (at leastone and half year) without extra expenses and regulatory risks/issues,as the case with animal derived products. Further, cells fromLeishmania-Bank do not need to undergo stringent tests for safety,including tests that screen for infectious agents. So, the costsassociated with use of such cells will increase much. This is not inprior art.

EXAMPLE-12

Determination of Infectivity of Promastigotes:

Previous studies suggest that factors such as culture medium (Neal R A,1984. Leishmania major: culture media, mouse strains, and promastigotesvirulence and infectivity. Exp. Parasitol. 57 (3): 269-273; O'Daly J Aand Rodriguez M B, 1988. Differential growth requirements of severalLeishmania spp. In chemically defined culture media. Acta Trop. 45(2):109-126; Merlen T, Sereno D, Brajon N, Rostand F and Lemesre J L, 1999.Leishmania spp: completely defined medium without serum andmacromolecules (CDM/LP) for the continuous in-vitro cultivation ofinfective promastigotes forms. Am. J. Trop. Med. Hyg. 60: 41-50; Dey T,Afrin F, Anam K and Ali N, 2002. Infectivity and virulence of Leishmaniadonovani promastigotes: a role for media, source, and strain ofparasite. J. Eukaryote Microbiol. 49 (4): 270-274), medium of primaryisolation (Neal R A, 1984. Leishmania major: culture media, mousestrains, and promastigotes virulence and infectivity. Exp. Parasitol. 57(3): 269-273), growth phases, number of sub-passages (Giannini M S,1974. Effects of promastigotes growth, frequency of subculture, and hostage on promastigotes—initiated infections with Leishmania donovani inthe golden hamster. J. Protozool. 21 (4), 521-527; Semprevivo L H,Yusuf, J N various laboratory procedures. Zeitschrift fur Parasitenkunde65, 43-51; Nolan T J and Herman R, 1985. Effects of long-term in vitrocultivation on Leishmania donovani promastigotes. J Protozool. 32(1):70-75), animal's species/strain and their age, sex, inoculum size (MbatiP A, Abok K, Orago A S, Anjili C O, Githure J I, Kagai J M and Koech DK, 1994. Establishment of an appropriate inoculum dose of Leishmaniadonovani promastigotes required to establish a visceral infection inlaboratory animal rodent models. Afr. J. Health Sci. 1(4): 165-168),route of inoculation etc affect the establishment of parasites in animalmodel. Infectivity studies showed that L. major promastigotes grown inSchneider's medium maintained infectivity to BALB/c mice throughout theperiod of cultivation. Infectivity to CD1 strain mice was progressivelylost. Promastigotes grown in blood agar medium, however, lostinfectivity to both strains of mice at a faster rate than promastigotesgrown in Schneider's medium (Neal R A, 1984. Leishmania major: culturemedia, mouse strains, and promastigotes virulence and infectivity. Exp.Parasitol. 57(3): 269-73). Schlein & Jacobson (Schlein Y and Jacobson RL, 1994. Mortality of Leishmania major in Phlebotomus papatasi caused byplant feeding of the sand flies. Am. J. Trop. Med. Hyg. 50:20-27)observed that haemoglobin or blood in the growth medium of L. majorinhibited the formation of infective promastigotes. Inoculation of micewith stationery-phase parasites from control medium caused infections in20/29 mice, compared to 3/20 mice injected with parasites grown withrabbit hemoglobin.

Hence, for developing a new medium for continuous cultivation ofLeishmania promastigotes, it is worthwhile to check the length oftime/number of sub-passage that could maintain the infectivity. Thefreshly transformed Leishmania promastigotes gradually lose theirinfectivity due to prolonged in vitro cultivation.

To determine the infectivity of promastigotes cultured in thisnutritionally changed/modified growth environment having plant originbased supplement, 1 (VL) primary isolate was successively sub-passagedduring log phase in LGPY medium supplemented with 2 different plants'extract (20%, v/v) whist LGPY with peptone of animal origin mediumsupplemented with 10% FCS was taken as control.

Promastigotes of stationery phase from both the media groups wereharvested by centrifugation at 2500 rpm for 20 minutes at 4° C. andwashed with sterile Locke's solution (pH 7.2). Rosettes of promastigoteswere dispersed by agitating through laboratory shaker. Inbred maleBALB/c mice (6 to 8 week old, weighing about 20 g+2 g) were distributedinto two groups of 3 animals in each for experiment. Promastigotes wereinoculated (1×10⁸ cells/animal) intraperitoneally from sub-passagenumber 8. Animals were maintained on commercial pellet food and givenwater ad libitum.

To determine the infectivity of promastigotes in spleen, animals fromboth groups were sacrificed in 3 months. Spleen was removed aseptically.Small pieces (minced) of spleen were inoculated in BHI Agar with 30%defibrinated rabbit blood (slant of ca. 3-5 ml) having Locke's solutionas overlay (ca. 0.5-1 ml) and Schneider's insect medium with 20% FCS and10% rabbit blood. Inoculated vials were incubated at 24° C.±1° C. andexamined for promastigotes after 4-5 days followed by every 1-2 daysinterval up to 21 days to say negative. Impression smears from the cutsurfaces of the spleen were prepared and fixed with methanol. Smear wasmicroscopically examined after staining with Giemsa for presence ofamastigotes.

Spleen was found positive for Leishmania parasites i.e. presence ofamastigotes/promastigotes. These results demonstrate, unquestionably,that the promastigotes forms of the visceral leishmaniasis as obtainedaccording to the invention are infectious in vivo means able totransform into amastigotes forms, survive, and multiply intracellularly,and retain infectivity in susceptible animal after certain (at least 8)successive sub-passaging.

For parasites causing both cutaneous and visceral leishmaniasis, Merlenet al. (Merlen T, Sereno D, Brajon N, Rostand F and Lemesre J L, 1999.Leishmania spp: completely defined medium without serum andmacromolecules (CDM/LP) for the continuous in-vitro cultivation ofinfective promastigotes forms. Am. J. Trop. Med. Hyg. 50: 41-50) foundthat the absence of serum, proteins, and peptides in their completelydefined culture medium (CDM/CP) did not markedly change their in vitroinfectivity for resident mouse macrophages and their virulence inanimals compared with parasites cultivated in non defined medium.Infectivity studies in hamsters and BALB/c mice showed thatpromastigotes isolated in biphasic M199 with 10% FCS were several foldsmore infective than those obtained from M199 with 10% FCS. The mediumused for the conversion of amastigotes to promastigotes plays a majorrole in determining the infectivity of the freshly transformed L.donovani promastigotes in hamsters and BALB/c mice (Dey T, Afrin F, AnamK and Ali N, 2002. Infectivity and virulence of Leishmania donovanipromastigotes: a role for media, source, and strain of parasite. J.Eukaryot. Microbiol. 49(4): 270-274).

EXAMPLE-13

Determination of Capability for Transformation of Amastigotes toPromastigotes:

The medium suitable for maintenance of promastigotes may not benecessarily suitable for primary isolation of promastigotes.

Hence, to determine the capability for transformation of amastigotes topromastigotes clinical material was inoculated in LGPY medium with 20%different plants' extract (code no. 5, 16, 26, 29, 44, 48). Inoculatedculture was placed in B.O.D incubator at 25° C.±1° C. After every 1-2days, a wet smear was examined over 3-4 weeks under the light microscopefor presence of motile promastigotes before considered as a negative.Promastigotes were found in this culture medium. Hence, the medium isproved to be capable for transformation of amastigotes to promastigotes.

EQUIVALENTS

While this disclosures has been described and illustrated using certainspecific embodiments, it will be understood that further modificationsmay be made and this application covers any variations, uses, oradaptations of the disclosed embodiments following, in general, theprinciples/processes of the invention and including such departures fromthe present disclosure as come within known or customary practice withinthe art to which the invention pertains and as may be applied to thefeatures herein and as follows in the spirit or scope of the appendedclaims.

To avoid detail not necessary to enable those skills of art to practicethe invention, the description omits certain information known to thoseskilled in the art. The preceding detailed description as well as claimsare, therefore, not to be taken in a limiting sense.

All publications, patents and patent applications mentioned in thisspecification are indicative of the level of skill of those skilled inthe art to which this invention pertains, and are herein incorporated byreference to the same extent as if each individual publication, patentor patent application was specifically and individually indicated to beincorporated herein by reference.

We claim:
 1. A culture medium for cultivation of Leishmaniapromastigotes, comprising at least one plant extract obtained from aplant selected from the group consisting of Citrus maxima, Thevetiaperuviana, Croton bonplandianum, Benincasa hispida, Dolichos lablab,Calotropis procera, Colocasia esculenta, Amorphophallus paeoniifolius,Coccinia cordifolia, Amaranthus dubius, Citrus aurantifelia, Cucurbitapepo, Sesbania sesban, Solanum melongena, Abutilon indicum, Trianthemaportulacastrum, and Basella alba wherein said culture medium isserum-free or animal product-free; and an inorganic salt selected fromthe group consisting of sodium chloride, potassium chloride, potassiumdihydrogen phosphate and calcium chloride.
 2. The culture medium asclaimed in claim 1, further comprising an organic constituent comprisingglucose and yeast extract.
 3. The culture medium as claimed in claim 1,wherein the inorganic salts are in the following amounts: SodiumChloride  8 g/1 Potassium Chloride 200 mg/1 Potassium dihydrogenphosphate 300 mg/1 and Calcium chloride 200 mg/1, and further comprising 5 g/1 of glucose,  5 g/1 of yeast, and 10 g/1 of Peptone/hydrolysateand Plant extract.


4. The culture medium as claimed in claim 3, wherein said plant extractis present in 5 to 20% by weight.
 5. The culture medium as claimed inclaim 1, for the transformation of amastigotes to promastigotes.
 6. Aculture medium for cultivation of Leishmania promastigotes, comprisingat least one plant extract obtained from a plant selected from the groupconsisting of Citrus maxima, Thevetia peruviana, Croton bonplandianum,Benincasa hispida, Dolichos lablab, Calotropis procera, Colocasiaesculenta, Amorphophallus paeoniifolius, Coccinia cordifolia, Amaranthusdubius, Citrus aurantifelia, Cucurbita pepo, Sesbania sesban, Solanummelongena, Abutilon indicum, Trianthema portulacastrum, and Basella albawherein said culture medium is serum-free or animal product-free; and anorganic constituent comprising glucose and yeast extract.
 7. The culturemedium as claimed in claim 6, further comprising an inorganic saltselected from the group consisting of sodium chloride, potassiumchloride, potassium dihydrogen phosphate and calcium chloride, whereinthe inorganic salts are in the following amounts: Sodium Chloride 8 g/lPotassium Chloride 200 mg/l Potassium dihydrogen phosphate 300 mg/l, andCalcium chloride 200 mg/l, and further comprising 5 g/l of glucose, 5g/l of yeast extract, and 10 g/l of Peptone/hydrolysate and Plantextract.
 8. The culture medium as claimed in claim 7, wherein said plantextract is present in 5 to 20% by weight.
 9. The culture medium asclaimed in claim 6, for the transformation of amastigotes topromastigotes.